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Serum leptin levels in patients with nonalcoholic steatohepatitis
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2000 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 95, No. 12, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(00)02090-6
Serum Leptin Levels in Patients With Nonalcoholic Steatohepatitis Ahmet Uygun, M.D., Abdurrahman Kadayifci, M.D., Zeki Yesilova, M.D., Ahmet Erdil, M.D., Halil Yaman, M.D., Mendane Saka, Ph.D., M. Salih Deveci, M.D., Sait Bagci, M.D., Mustafa Gulsen, M.D., Necmettin Karaeren, M.D., and Kemal Dagalp, M.D. Departments of Gastroenterology, Biochemistry, and Pathology, Gulhane Military Medical Academy, Ankara, Turkey
OBJECTIVE: Leptin is a peptide hormone that mainly regulates food intake and energy expenditure of human body. A close correlation between serum leptin levels and the percentage of body fat stores is well known. Nonalcoholic steatohepatitis (NASH) is a common disorder which causes serum liver enzyme elevation. In this study, the serum leptin levels were investigated in patients with NASH to determine a possible role in the pathogenesis and in patients with chronic viral hepatitis to ascertain the effect of hepatic inflammation on serum leptin level. METHODS: Forty-nine patients (38 men, 11 women) with NASH diagnosed by biopsy, 32 patients with biopsy-proven chronic viral hepatitis (21 men and 11 women), and 30 healthy adults (17 men, 13 women) enrolled in the study. Fasting blood samples were obtained, and serum leptin levels were measured by ELISA. Body mass index (BMI) was calculated for all subjects, and serum insulin, C-peptide, and lipoprotein levels were also detected. RESULTS: The mean serum leptin levels (⫾SEM) were 6.62 ⫾ 0.71, 4.24 ⫾ 1.0, and 4.02 ⫾ 0.46 ng/ml in NASH, chronic hepatitis, and the control group, respectively. Mean serum leptin level in the NASH group was significantly higher than those in the other groups tested. BMI was also slightly higher in the NASH group when compared to the other groups (26.7 ⫾ 0.3, 23.7 ⫾ 0.6, and 24.6 ⫾ 0.3, respectively). There was a significant correlation between BMI and serum leptin levels when all the subjects were evaluated together (NASH, hepatitis, and control groups, r ⫽ 0.337, p ⫽ 0.012) but not in the NASH group when evaluated alone (r ⫽ 0.238, p ⫽ 0.1). Of the predisposing factors for NASH, obesity was observed in 24% of patients and hyperlipidemia in 67%. Serum cholesterol and triglyceride levels were significantly higher in the NASH group than those in controls (p ⬍ 0.05). It has been detected that most of these patients consumed high amounts of fat in their dietary habits. CONCLUSIONS: The serum leptin levels were significantly higher in patients with NASH, while they were not affected by chronic hepatitis. This elevation is out of proportion to BMI of these patients and may be related to hyperlipidemia
in most. Elevated serum leptin levels, therefore, may promote hepatic steatosis and steatohepatitis. (Am J Gastroenterol 2000;95:3584 –3589. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION Leptin is a recently isolated protein encoded by the ob gene, which is an adipocyte-specific gene (1). It was suggested that leptin regulates body weight by decreasing food intake and increasing energy expenditure (2). It is mainly effective through the central hypothalamic mechanism. Although little has been uncovered about leptin and its regulation in humans, it seems that it plays an essential role in the pathophysiology of obesity (3). Leptin is expressed exclusively in adipose tissue, and the serum leptin levels correlate well, and to a close degree, with the percentage of body fat and body mass index (BMI) (4, 5). A number of studies have shown that obese individuals have higher levels of circulating leptin compared to lean subjects (2– 6). This elevation is mainly caused by a receptor resistance to the effects of leptin in obesity (7). Moreover, serum leptin levels are higher in women than in men, secondary to body fat composition and sex hormones (8). Nonalcoholic steatohepatitis (NASH) defines liver injury due to the accumulation of fat in hepatocytes. The etiology of NASH is not known completely, but it is found more frequently in patients with obesity, non–insulin-dependent diabetes mellitus, and hyperlipidemia (9). However, it may be seen in some patients without any detectable predisposing factor (10). In addition, the pathogenesis of NASH remains unclear. Increased synthesis of fatty acids in the liver, increased delivery of free fatty acids to the liver, and decreased -oxidation of free fatty acids may cause the accumulation of fat in the liver (9). Therefore, fat in hepatocytes causes cellular dysfunction and may damage liver parenchyma. The close relationship of leptin with adipose tissue and fat stores of the body suggests its possible involvement in the etiology and pathogenesis of NASH. On the other hand, elevated serum leptin levels determined in patients with liver cirrhosis in recent studies suggest that the
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Table 1. The Demographic Variables and Body Composition in Groups Variable
NASH
Hepatitis
Control
p
Gender (M/F) Age, yr (range) Weight, kg (range) Height, cm (range) BMI, kg/m2 (range)
38/11 39.3 ⫾ 1.7 (26–65) 78.8 ⫾ 1.37 (59–99) 171 ⫾ 1.2 (150–190) 26.73 ⫾ 0.3 (21.67–33.22)
21/11 41.7 ⫾ 2.8 (29–61) 68.7 ⫾ 1.37 (46–91) 170 ⫾ 1.6 (148–186) 23.75 ⫾ 0.6 (19–29.37)
17/13 37.1 ⫾ 1.3 (23–58) 71.5 ⫾ 1.8 (49–89) 170 ⫾ 1.4 (156–187) 24.59 ⫾ 0.3 (20.7–29.06)
NS NS S NS S
Data are mean ⫾ SEM. NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
alterations in liver functions may influence the levels of circulating leptin (11–13). However, the underlying mechanism and pathophysiological significance of this result has not yet been interpreted satisfactorily. It is a possibility that impaired hepatic clearance in cirrhosis may simply cause the elevation in serum leptin level. However, inflammatory response in damaged hepatocytes may promote a signaling effect on adipose tissue via known or unknown leptin’s secretogogues. We are aware that reports are not available in the medical literature with regards to circulating leptin levels in patients with NASH and in patients with chronic hepatitis without cirrhosis. Thus, the role of leptin on NASH and/or the effects of hepatic inflammation on serum leptin levels are obscure at present. The aim of this study is to evaluate the serum leptin levels in patients with NASH and chronic hepatitis without cirrhosis, in order to contribute to the clarification of the mechanism in steatohepatitis.
MATERIALS AND METHODS A total of 49 patients with NASH and 32 patients with chronic viral hepatitis were enrolled in the study, in addition to 30 healthy persons as controls (Table 1). NASH patients were admitted or referred to our department with incidentally found liver enzyme elevations. For the diagnoses of NASH and to rule out other possible liver diseases, all patients with NASH underwent a detailed clinical and laboratory evaluation, including liver function tests, hepatitis markers, and autoantibodies. Patients with suspected acute or chronic viral hepatitis and autoimmune hepatitis were not included in this study group. All subjects underwent an upper abdominal ultrasonography. NASH was definitively diagnosed in all patients by histopathological examination following liver biopsy. The combination of hepatocellular steatosis, ballooning and disarray, acinar or portal inflammation, and fibrosis in histopathological examinations was graded as suggested by the necroinflammatory grading system for steatohepatitis and defined as grade 1 (mild), grade 2 (moderate), and grade 3 (severe) steatohepatitis (14). Alcohol consumption was absent in 41 patients and negligible (⬍100 g of ethanol per month) in the remaining 8 patients. Patients with previous or current history of malignant disease; pituitary, adrenal, and pancreatic disease; prolonged use of corticosteroids or sex hormones; or history of GI surgical procedures were excluded. The chronic hepatitis group consisted of 18 type B and 14
type C patients. All of the hepatitis patients had ALT and AST elevations during the last 6 months and had not received interferon or any other antiviral treatment within this period. The histopathological findings showed varying degrees of portal inflammation and parenchymal injury without advanced fibrosis and/or cirrhosis. Patients with obvious signs of acute or chronic systemic illnesses except for viral hepatitis were not included in the study. Serum insulin, C-peptide, and lipoprotein levels were analyzed in addition to other biochemical tests in all patients and controls. All patients were evaluated on an outpatient basis. BMI was calculated as weight (in kilograms) divided by the square of height (in meters) in all patients and controls. Obesity was defined as BMI ⬎27.8, for men and ⬎27.3, for women (15). In addition, patients with a BMI ⬎30 were defined as manifest obese. A dietitian administered a food frequency questionnaire to all participants to detect estimated dietary intake. In this questionnaire, the participants were asked about the consumption of all nutritional elements within the last 7 days. Then, daily calorie intake and proportional distribution of lipid, protein, and carbohydrates in daily calorie consumption were calculated by using a food composition scale. Those subjects who provided more than 30% of daily calorie intake from lipids were accepted as high fat consumers (16). Informed written consent was obtained for each subject. All blood samples were collected in the morning after an overnight fast, and 2 ml of serum samples were stored at ⫺30°C until assayed for leptin. Serum leptin level was measured by a commercially available ELISA kit (The DSL-10 –23100 Human Leptin Enzyme-Linked Immunosorbent Kit; Diagnostic Systems Laboratories, Webster, TX). The principle of the test is an enzymatically amplified, two-step, sandwich-type immunoassay. In this assay, standards, controls, and unknown serum or plasma samples are incubated in microtitration wells, which have been coated with anti– human leptin antibody. After a two-step incubation and washing procedure, an acidic stopping solution is added, and the degree of enzymatic turnover of the substrate is determined by chromatography. The minimum limit of detection was 0.05 ng/ml. The intra-assay and interassay coefficients of variation were below 6.2 and 5.3%, respectively (n ⫽ 12). All results are expressed as mean ⫾ SEM. The mean leptin levels, BMI, and other serum parameters among groups were compared first by one-way Anova analysis of variance. The unpaired Student t test was used for dual
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Table 2. The Results of Main Biochemical Tests in Groups Group Tests
NASH
Hepatitis
Controls
p
Leptin (ng/ml) C-peptide (ng/ml) Insulin (ng/ml) ALT (u/L) AST (u/L) Glucose (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)
6.62 ⫾ 0.7 2.89 ⫾ 0.1 19.97 ⫾ 1.2 69.44 ⫾ 4.2 49.34 ⫾ 1.96 94.04 ⫾ 2.9 231.26 ⫾ 5.6 205.1 ⫾ 13.3 157.2 ⫾ 5.4 43.8 ⫾ 1.2
4.24 ⫾ 1.0 2.81 ⫾ 0.1 21.78 ⫾ 1.2 54.46 ⫾ 4.0 59.03 ⫾ 3.8 88.34 ⫾ 1.9 190.75 ⫾ 8.1 135.93 ⫾ 7.5 126.5 ⫾ 4.3 41.2 ⫾ 1.4
4.02 ⫾ 0.4 2.69 ⫾ 0.1 21.21 ⫾ 0.8 26.63 ⫾ 1.2 30.83 ⫾ 0.9 86.66 ⫾ 1.8 183.0 ⫾ 5.6 116.83 ⫾ 10.1 120.0 ⫾ 5.7 42.7 ⫾ 1.0
S NS NS S S NS S S S NS
Data are mean ⫾ SEM. HDL ⫽ high density lipoprotein; NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
comparisons between groups. The correlation between leptin and BMI was given by the Pearson correlation coefficient (r). The unpaired Student t test and the 2 test were used to compare the differences in demographic values between the groups. All analyses were two tailed and were conducted using a computer-based statistics software (SPSS for Windows 8.0, 1997, SPSS, Chicago, IL). A p value of less than 0.05 was accepted as statistically significant.
RESULTS The results of main parameters studied in groups are summarized in Table 2. The main predisposing factors for NASH were hyperlipidemia in 33 patients, obesity in 12 patients, and diabetes mellitus in 3 patients. Only 3 of the obese patients fit the definition of manifest obesity. All the diabetics and 7 of the obese patients had hyperlipidemia as well. Therefore, 38 patients had one or more predisposing factors for NASH, whereas the remaining 11 had no detectable predisposing cause. Four of the subjects in the control group were also obese, and 8 patients had hyperlipidemia. Despite the fact that about 75% of NASH patients were men in our group, since most of our patients were military personnel, sex distribution of patients in NASH and control group was not significantly different (2 ⫽ 2.91, p ⬎ 0.05). Serum leptin levels were found to be significantly higher in patients with NASH (6.62 ⫾ 0.7 ng/ml) as compared with both the control group (4.02 ⫾ 0.4 ng/ml, p ⫽ 0.011) and the chronic hepatitis group (4.24 ⫾ 1 ng/ml, p ⫽ 0.014; Fig. 1). There was no significant difference between mean levels of serum leptin of chronic hepatitis subjects and those of control subjects (p ⫽ 0.82). As expected, serum leptin levels were significantly higher in women subjects compared with those of men in all groups (p ⬍ 0.05; Table 3). For this reason, all comparisons were also made between men and women separately. Both men and women in the NASH group had higher mean serum leptin levels than did the men and women subjects of the control and chronic hepatitis groups (p ⬍ 0.05; Table 3). There was a positive correlation between BMI and leptin values for the combined groups (NASH, hepatitis, and con-
trol, r ⫽ 0.337, p ⫽ 0.012). When groups were analyzed separately, the correlation was also significant in the control group (r ⫽ 0.430, p ⫽ 0.018) and the hepatitis group (r ⫽ 0.441, p ⫽ 0.001), but not in the NASH group (r ⫽ 0.238, p ⫽ 0.1). Because the proportion of obese patients was rather low in the study population, for determining the relationship between obesity and leptin more clearly, the NASH patients were divided into two groups by using a BMI cutpoint score of 26. Thus, patients with a BMI of ⬎26 were defined as overweight. There were 20 patients with a BMI of ⬍26, and there were 29 patients with a BMI ⱖ26. The comparison of leptin values between these two groups was not significant, and the mean values were very close (Table 4). The serum leptin values were statistically higher in each of the two NASH groups (BMI ⬍ 26 or ⱖ26) than for those in the control group (p ⬍ 0.05 for both). These analyses revealed that the leptin values in NASH patients were higher in every way than were those of control subjects, without any significant effect for BMI. Histopathological examinations revealed that 29 of the
Figure 1. The serum leptin levels in nonalcoholic steatohepatitis patients, chronic hepatitis patients, and controls (bar graphic with 95% confidence interval for the mean).
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Table 3. Leptin and BMI Results of Groups According to Gender Men
Women
Variable
NASH
Hepatitis
Controls
Number (n) Leptin (ng/ml) BMI (kg/ml)
38 5.33 ⫾ 0.6 26.62 ⫾ 0.3
21 2.89 ⫾ 0.9 23.6 ⫾ 0.7
17 3.04 ⫾ 0.4 24.61 ⫾ 0.3
p
NASH
Hepatitis
Controls
p
S S
11 11.09 ⫾ 1.8 27.13 ⫾ 0.7
11 6.83 ⫾ 2.1 23.93 ⫾ 1.0
13 5.30 ⫾ 0.8 24.56 ⫾ 0.7
S S
S ⫽ significant at a p value of ⬍0.05.
NASH patients had grade 1 necroinflammatory activity, whereas 17 had grade 2 activity, and only 3 had grade 3 steatohepatitis. The mean level of serum leptin was 5.81 ⫾ 1.1 ng/ml in grade 1 patients and was 7.79 ⫾ 0.5 and 7.22 ⫾ 1.2 ng/ml in grade 2 and 3 patients, respectively. Because there were only three patients with grade 3 inflammation, they were included with the group 2 patients for statistical analyses. The mean serum leptin levels of the 20 patients with grade 2 and 3 hepatic inflammatory activity was significantly higher when compared with those of the 29 patients with grade 1 steatohepatitis (p ⫽ 0.018). We detected from the food frequency questionnaire that 42 of the patients with NASH were high fat consumers, whereas this was the case for only 7 of the controls. From the NASH patients with hyperlipidemia, 31 (93.3%) were also in the high fat consumer group. The C-peptide and insulin levels did not differ among groups (p ⬎ 0.05), but a positive correlation was found between serum leptin and insulin levels of NASH patients (r ⫽ 0.297, p ⫽ 0.038). The serum ALT, AST, glucose, total cholesterol, triglyceride, and low density lipoprotein (LDL) cholesterol levels were statistically higher in NASH patients than in controls (p ⬎ 0.05). However, none of these parameters displayed significant correlation with serum leptin levels in NASH patients.
DISCUSSION In this study, significantly high levels of serum leptin in NASH patients were found, which to our surprise, did not correlate with the BMI levels of the same group. On the other hand, a positive correlation between BMI and serum leptin levels was determined for the hepatitis subjects and Table 4. The Results of BMI, Leptin, and Main Biochemical Tests in NASH Patients With Normal Weight and Overweight Tests
Overweight (BMI ⱖ 26)
Normal Weight (BMI ⬍ 26)
p
Number (n) BMI (kg/m2) Leptin (ng/ml) C-peptide (ng/ml) Insulin (ng/ml) ALT (u/L) AST (u/L) Glucose (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl)
29 28.1 ⫾ 0.3 6.64 ⫾ 1.0 2.93 ⫾ 0.1 20.7 ⫾ 1.3 72.8 ⫾ 4.4 48.7 ⫾ 1.9 96.3 ⫾ 3.1 233.7 ⫾ 5.7 204.4 ⫾ 13.3
20 24.7 ⫾ 0.2 6.59 ⫾ 1.0 2.85 ⫾ 0.1 19.1 ⫾ 1.1 67.6 ⫾ 4.1 49.8 ⫾ 1.9 93.9 ⫾ 3.4 229.1 ⫾ 5.6 206.1 ⫾ 13.3
S NS NS NS NS NS NS NS NS
Data are mean ⫾ SEM. NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
the control groups. The above results suggest no difference between normal and overweight NASH patients with regard to serum leptin levels. Our present findings contrast with previous reports suggesting higher levels of leptin in obese patients (2– 6). On the one hand, obesity is closely correlated with elevated levels of serum leptin; therefore, the majority of NASH patients may be expected to have high levels of serum leptin. On the other hand, the majority of our study patients displaying high levels of serum leptin were nonobese. Moreover, only three patients had a BMI above 30, which is the threshold for manifest obesity. This is probably because most of our patients were military personnel, among whom obesity is rarely seen. In fact, this situation may be considered to have been an advantage in displaying the role of leptin more clearly in NASH patients. It is an expected finding that leptin may increase in manifest obese patients with NASH. In such a patient group, it is difficult to explain whether serum leptin elevation is simply a consequence of obesity or whether it is really related to NASH. However, our study shows that serum leptin levels are increased in nonobese NASH patients and that it is impossible to explain serum leptin elevations in terms of obesity in the study group. Most of the NASH patients in this study exhibited hyperlipidemia. Serum total cholesterol, triglyceride, and LDL cholesterol levels of NASH patients were significantly elevated compared to controls. Lack of a familial hyperlipidemia history, absence of tendon xanthomas in examination, and no clinical disorders associated with secondary hyperlipidemia, except diabetes mellitus in three patients, suggested that in most of our patients, their lipoprotein concentrations are mainly influenced by their diet. It was determined from the food frequency questionnaire that nearly all the patients with hyperlipidemia were high-fat consumers in the study. This observation makes us consider the possibility that hyperlipidemia and a high-fat diet may induce important changes in leptin expression in adipose tissue without a significant increase in body fat content. In line with this suggestion, it was reported in a recent study that serum leptin levels were found elevated out of proportion to the BMI in patients with familial hyperlipidemia (17). The effect of a high-fat diet on serum leptin level is controversial in that a short-term high-fat diet may lower serum leptin levels (18, 19), but a long-term high-fat diet or habitual high-fat consumption is usually associated with increased serum leptin levels (20, 21). On the other hand, it is well known that food intake is the major promoting factor
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for leptin secretion. According to our results, like other previous studies, it can be suggested that a continuous high-fat diet and hyperlipidemia may result in high levels of serum leptin without a marked increase in BMI and manifest obesity. In a few recent studies, it has been demonstrated that serum leptin levels are increased in patients with liver cirrhosis (11–13). These studies confirm the suggestions that liver may play an important role on leptin metabolism and contribute to the regulation of circulating leptin levels. Neither the pathophysiological significance of this elevation nor the underlying exact mechanisms are clear at present. It has been suggested that hepatic failure in cirrhosis may cause this elevation by simply altering metabolism and clearance of leptin. In this study, no difference was determined between serum leptin levels of patients with chronic viral hepatitis and those of controls. Clinical, biochemical, or histopathological signs suggesting hepatic failure in hepatitis patients were not observed, and all the participants were in the uncirrhotic phase of the disease. At this point, our study confirms that leptin elevation in liver cirrhosis seems to be related to hepatic failure rather than to chronic inflammation or host immune response related to hepatic cell injury. On the other hand, the similar serum leptin levels in chronic viral hepatitis patients and controls point out that the increased serum leptin levels in NASH do not result simply from the liver damage. Whether serum leptin elevation has a metabolic effect in the development of steatosis and steatohepatitis is not clear at present. One of the important results of the study is that higher serum leptin levels were detected in patients with grade 2 and 3 steatohepatitis, compared with the grade 1 patients. This finding suggests that increased serum leptin level may be involved in the pathogenesis of steatohepatitis. However, this study is not enough to explain a possible relationship between leptin and hepatic fat accumulation and hepatic injury. It has been suggested that leptin may contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes so as to promote increased intracellular fatty acids (22). Moreover, in a later stage, leptin may cause hepatic steatosis to turn into steatohepatitis by amplifying selected proinflammatory responses (22). This explanation mainly is based on the effect of insulin on hepatic fat metabolism. In this study, an elevation of serum insulin level in NASH patients, in comparison to the control group, was not detected, but a positive significant correlation was determined between serum leptin and insulin levels, which supports the latter suggestion. It has been suggested that leptin substitution reverses insulin resistance and hepatic fat accumulation in some animal models with congenital lipodystrophy and leptin deficiency (23). However, this is not in question in the situation going on with increased leptin levels that has been shown to promote insulin resistance and probably intracellular fatty acids in hepatocytes (22). In summary, the data suggest that serum leptin level is
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increased in NASH patients inproportional to BMI. The high serum leptin levels may be secondary to hyperlipidemia in a significant number of these patients. Elevated serum leptin levels may therefore promote hepatic steatosis and steatohepatitis by insulin secretion and/or proinflammatory activation. However, further studies are needed to determine more clearly the role of leptin on hepatic steatosis and steatohepatitis.
Reprint requests and correspondence: Ahmet Uygun, M.D., GATA Lojmanları, Rieder Pasa, Apartment 5, Etlik/Ankara, Turkey. Received Sep. 14, 1999; accepted June 29, 2000.
REFERENCES 1. Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372:425–32. 2. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763–70. 3. Considine RV, Sinha MK, Heiman ML, et al. Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 1996;334:292–5. 4. Mantzoros CS, Moschos S, Avramopoulos I, et al. Leptin concentrations in relation to body mass index and the tumour necrosis factor-␣ system in humans. J Clin Endocrinol Metab 1997;82:3408 –13. 5. Blum WF, Englaro P, Hanitsch S, et al. Plasma leptin levels in healthy children and adolescents: Dependence on body mass index, body fat mass, gender, pubertal stage, and testosterone. J Clin Endocrinol Metab 1997;82:2904 –10. 6. Lo¨nnquist F, Arner P, Nordfors L, et al. Overexpression of the obese (ob) gene in adipose tissue of human obese subjects. Nat Med 1995;1:950 –3. 7. Friedman JM. Leptin, leptin receptors and the control of body weight. Eur J Med Res 1997;2:7–13. 8. Hickey MS, Israel RG, Gardiner SN, et al. Gender difference in serum leptin levels in humans. Biochem Mol Med 1996; 59:1– 6. 9. Shelh SG, Gordon FD, Chopra S. Nonalcoholic steatohepatitis. Ann Intern Med 1997;126:137– 45. 10. Bacon BR, Farahvash MJ, Jannery CG, et al. Nonalcoholic steatohepatitis; an expanded clinical entity. Gastroenterology 1994;107:1103–9. 11. Mccullough AJ, Bugianesi E, Marchesini G, et al. Genderdependent alterations in serum leptin in alcoholic cirrhosis. Gastroenterology; 1998;115:947–53. 12. Shimizu H, Kakizaki S, Tsuchiya T, et al. An increase of circulating leptin in patients with liver cirrhosis. Int J Obes Relat Metab Disord 1998;22:1234 – 8. 13. Henriksen JH, Holst JJ, Moller S, et al. Increased circulating leptin in alcoholic cirrhosis: relation to release and disposal. Hepatology 1999;26:1818 –24. 14. Brunt AM, Janney CG, Di Bisceglie AM, et al. Nonalcoholic steatohepatitis. A proposal for grading and staging the histological lesions. Am J Gastroenterol 1999;94:2467–74. 15. NIH Technology Assessment Conference Panel. Methods for voluntary weight loss and control. Ann Intern Med 1992;116: 942–9.
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16. Pao EM, Cypel YS. Estimation of dietary intake. In: Ziegler EE, Filer LJ, eds. Present knowledge in nutrition, 7th ed. Washington, DC: ILSI Press, 1996:498 –507. 17. Jacobson MS, Yoon DJ, Frank GR. Serum leptin is elevated out of proportion to the body mass index in adolescent females with familial combined hyperlipidemia (FCH). Clin Pediatr 1999;38:49 –53. 18. Schrauwen P, Van Marken Lichtenbelt WD, Westerterp KR, et al. Effect of diet composition on leptin concentration in lean subjects. Metabolism 1997;46:420 – 4. 19. Weigle DS, Duell PB, Connor WE, et al. Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels. J Clin Endocrinol Metab 1997;82:561–5.
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20. Lovejoy JC, Windhauser MM, Rood JC, et al. Effect of a controlled high-fat versus low-fat diet on insulin sensitivity and leptin levels in African-American and Caucasian women. Metabolism 1998;47:1520 – 4. 21. Cooling J, Barth J, Blundell J. The high-fat phenotype: Is leptin involved in the adaptive response to a high fat (high energy) diet? Int J Obes Relat Metab Disord 1998;22: 1132–5. 22. Kaplan LM. Leptin, obesity and liver disease. Gastroenterology 1998;115:997–1001. 23. Shimomura I, Hammer RE, Ikemoto S, et al. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 1999;401:73– 6.
Vol. 95, No. 12, 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(00)02090-6
Serum Leptin Levels in Patients With Nonalcoholic Steatohepatitis Ahmet Uygun, M.D., Abdurrahman Kadayifci, M.D., Zeki Yesilova, M.D., Ahmet Erdil, M.D., Halil Yaman, M.D., Mendane Saka, Ph.D., M. Salih Deveci, M.D., Sait Bagci, M.D., Mustafa Gulsen, M.D., Necmettin Karaeren, M.D., and Kemal Dagalp, M.D. Departments of Gastroenterology, Biochemistry, and Pathology, Gulhane Military Medical Academy, Ankara, Turkey
OBJECTIVE: Leptin is a peptide hormone that mainly regulates food intake and energy expenditure of human body. A close correlation between serum leptin levels and the percentage of body fat stores is well known. Nonalcoholic steatohepatitis (NASH) is a common disorder which causes serum liver enzyme elevation. In this study, the serum leptin levels were investigated in patients with NASH to determine a possible role in the pathogenesis and in patients with chronic viral hepatitis to ascertain the effect of hepatic inflammation on serum leptin level. METHODS: Forty-nine patients (38 men, 11 women) with NASH diagnosed by biopsy, 32 patients with biopsy-proven chronic viral hepatitis (21 men and 11 women), and 30 healthy adults (17 men, 13 women) enrolled in the study. Fasting blood samples were obtained, and serum leptin levels were measured by ELISA. Body mass index (BMI) was calculated for all subjects, and serum insulin, C-peptide, and lipoprotein levels were also detected. RESULTS: The mean serum leptin levels (⫾SEM) were 6.62 ⫾ 0.71, 4.24 ⫾ 1.0, and 4.02 ⫾ 0.46 ng/ml in NASH, chronic hepatitis, and the control group, respectively. Mean serum leptin level in the NASH group was significantly higher than those in the other groups tested. BMI was also slightly higher in the NASH group when compared to the other groups (26.7 ⫾ 0.3, 23.7 ⫾ 0.6, and 24.6 ⫾ 0.3, respectively). There was a significant correlation between BMI and serum leptin levels when all the subjects were evaluated together (NASH, hepatitis, and control groups, r ⫽ 0.337, p ⫽ 0.012) but not in the NASH group when evaluated alone (r ⫽ 0.238, p ⫽ 0.1). Of the predisposing factors for NASH, obesity was observed in 24% of patients and hyperlipidemia in 67%. Serum cholesterol and triglyceride levels were significantly higher in the NASH group than those in controls (p ⬍ 0.05). It has been detected that most of these patients consumed high amounts of fat in their dietary habits. CONCLUSIONS: The serum leptin levels were significantly higher in patients with NASH, while they were not affected by chronic hepatitis. This elevation is out of proportion to BMI of these patients and may be related to hyperlipidemia
in most. Elevated serum leptin levels, therefore, may promote hepatic steatosis and steatohepatitis. (Am J Gastroenterol 2000;95:3584 –3589. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION Leptin is a recently isolated protein encoded by the ob gene, which is an adipocyte-specific gene (1). It was suggested that leptin regulates body weight by decreasing food intake and increasing energy expenditure (2). It is mainly effective through the central hypothalamic mechanism. Although little has been uncovered about leptin and its regulation in humans, it seems that it plays an essential role in the pathophysiology of obesity (3). Leptin is expressed exclusively in adipose tissue, and the serum leptin levels correlate well, and to a close degree, with the percentage of body fat and body mass index (BMI) (4, 5). A number of studies have shown that obese individuals have higher levels of circulating leptin compared to lean subjects (2– 6). This elevation is mainly caused by a receptor resistance to the effects of leptin in obesity (7). Moreover, serum leptin levels are higher in women than in men, secondary to body fat composition and sex hormones (8). Nonalcoholic steatohepatitis (NASH) defines liver injury due to the accumulation of fat in hepatocytes. The etiology of NASH is not known completely, but it is found more frequently in patients with obesity, non–insulin-dependent diabetes mellitus, and hyperlipidemia (9). However, it may be seen in some patients without any detectable predisposing factor (10). In addition, the pathogenesis of NASH remains unclear. Increased synthesis of fatty acids in the liver, increased delivery of free fatty acids to the liver, and decreased -oxidation of free fatty acids may cause the accumulation of fat in the liver (9). Therefore, fat in hepatocytes causes cellular dysfunction and may damage liver parenchyma. The close relationship of leptin with adipose tissue and fat stores of the body suggests its possible involvement in the etiology and pathogenesis of NASH. On the other hand, elevated serum leptin levels determined in patients with liver cirrhosis in recent studies suggest that the
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Table 1. The Demographic Variables and Body Composition in Groups Variable
NASH
Hepatitis
Control
p
Gender (M/F) Age, yr (range) Weight, kg (range) Height, cm (range) BMI, kg/m2 (range)
38/11 39.3 ⫾ 1.7 (26–65) 78.8 ⫾ 1.37 (59–99) 171 ⫾ 1.2 (150–190) 26.73 ⫾ 0.3 (21.67–33.22)
21/11 41.7 ⫾ 2.8 (29–61) 68.7 ⫾ 1.37 (46–91) 170 ⫾ 1.6 (148–186) 23.75 ⫾ 0.6 (19–29.37)
17/13 37.1 ⫾ 1.3 (23–58) 71.5 ⫾ 1.8 (49–89) 170 ⫾ 1.4 (156–187) 24.59 ⫾ 0.3 (20.7–29.06)
NS NS S NS S
Data are mean ⫾ SEM. NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
alterations in liver functions may influence the levels of circulating leptin (11–13). However, the underlying mechanism and pathophysiological significance of this result has not yet been interpreted satisfactorily. It is a possibility that impaired hepatic clearance in cirrhosis may simply cause the elevation in serum leptin level. However, inflammatory response in damaged hepatocytes may promote a signaling effect on adipose tissue via known or unknown leptin’s secretogogues. We are aware that reports are not available in the medical literature with regards to circulating leptin levels in patients with NASH and in patients with chronic hepatitis without cirrhosis. Thus, the role of leptin on NASH and/or the effects of hepatic inflammation on serum leptin levels are obscure at present. The aim of this study is to evaluate the serum leptin levels in patients with NASH and chronic hepatitis without cirrhosis, in order to contribute to the clarification of the mechanism in steatohepatitis.
MATERIALS AND METHODS A total of 49 patients with NASH and 32 patients with chronic viral hepatitis were enrolled in the study, in addition to 30 healthy persons as controls (Table 1). NASH patients were admitted or referred to our department with incidentally found liver enzyme elevations. For the diagnoses of NASH and to rule out other possible liver diseases, all patients with NASH underwent a detailed clinical and laboratory evaluation, including liver function tests, hepatitis markers, and autoantibodies. Patients with suspected acute or chronic viral hepatitis and autoimmune hepatitis were not included in this study group. All subjects underwent an upper abdominal ultrasonography. NASH was definitively diagnosed in all patients by histopathological examination following liver biopsy. The combination of hepatocellular steatosis, ballooning and disarray, acinar or portal inflammation, and fibrosis in histopathological examinations was graded as suggested by the necroinflammatory grading system for steatohepatitis and defined as grade 1 (mild), grade 2 (moderate), and grade 3 (severe) steatohepatitis (14). Alcohol consumption was absent in 41 patients and negligible (⬍100 g of ethanol per month) in the remaining 8 patients. Patients with previous or current history of malignant disease; pituitary, adrenal, and pancreatic disease; prolonged use of corticosteroids or sex hormones; or history of GI surgical procedures were excluded. The chronic hepatitis group consisted of 18 type B and 14
type C patients. All of the hepatitis patients had ALT and AST elevations during the last 6 months and had not received interferon or any other antiviral treatment within this period. The histopathological findings showed varying degrees of portal inflammation and parenchymal injury without advanced fibrosis and/or cirrhosis. Patients with obvious signs of acute or chronic systemic illnesses except for viral hepatitis were not included in the study. Serum insulin, C-peptide, and lipoprotein levels were analyzed in addition to other biochemical tests in all patients and controls. All patients were evaluated on an outpatient basis. BMI was calculated as weight (in kilograms) divided by the square of height (in meters) in all patients and controls. Obesity was defined as BMI ⬎27.8, for men and ⬎27.3, for women (15). In addition, patients with a BMI ⬎30 were defined as manifest obese. A dietitian administered a food frequency questionnaire to all participants to detect estimated dietary intake. In this questionnaire, the participants were asked about the consumption of all nutritional elements within the last 7 days. Then, daily calorie intake and proportional distribution of lipid, protein, and carbohydrates in daily calorie consumption were calculated by using a food composition scale. Those subjects who provided more than 30% of daily calorie intake from lipids were accepted as high fat consumers (16). Informed written consent was obtained for each subject. All blood samples were collected in the morning after an overnight fast, and 2 ml of serum samples were stored at ⫺30°C until assayed for leptin. Serum leptin level was measured by a commercially available ELISA kit (The DSL-10 –23100 Human Leptin Enzyme-Linked Immunosorbent Kit; Diagnostic Systems Laboratories, Webster, TX). The principle of the test is an enzymatically amplified, two-step, sandwich-type immunoassay. In this assay, standards, controls, and unknown serum or plasma samples are incubated in microtitration wells, which have been coated with anti– human leptin antibody. After a two-step incubation and washing procedure, an acidic stopping solution is added, and the degree of enzymatic turnover of the substrate is determined by chromatography. The minimum limit of detection was 0.05 ng/ml. The intra-assay and interassay coefficients of variation were below 6.2 and 5.3%, respectively (n ⫽ 12). All results are expressed as mean ⫾ SEM. The mean leptin levels, BMI, and other serum parameters among groups were compared first by one-way Anova analysis of variance. The unpaired Student t test was used for dual
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Table 2. The Results of Main Biochemical Tests in Groups Group Tests
NASH
Hepatitis
Controls
p
Leptin (ng/ml) C-peptide (ng/ml) Insulin (ng/ml) ALT (u/L) AST (u/L) Glucose (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl) LDL cholesterol (mg/dl) HDL cholesterol (mg/dl)
6.62 ⫾ 0.7 2.89 ⫾ 0.1 19.97 ⫾ 1.2 69.44 ⫾ 4.2 49.34 ⫾ 1.96 94.04 ⫾ 2.9 231.26 ⫾ 5.6 205.1 ⫾ 13.3 157.2 ⫾ 5.4 43.8 ⫾ 1.2
4.24 ⫾ 1.0 2.81 ⫾ 0.1 21.78 ⫾ 1.2 54.46 ⫾ 4.0 59.03 ⫾ 3.8 88.34 ⫾ 1.9 190.75 ⫾ 8.1 135.93 ⫾ 7.5 126.5 ⫾ 4.3 41.2 ⫾ 1.4
4.02 ⫾ 0.4 2.69 ⫾ 0.1 21.21 ⫾ 0.8 26.63 ⫾ 1.2 30.83 ⫾ 0.9 86.66 ⫾ 1.8 183.0 ⫾ 5.6 116.83 ⫾ 10.1 120.0 ⫾ 5.7 42.7 ⫾ 1.0
S NS NS S S NS S S S NS
Data are mean ⫾ SEM. HDL ⫽ high density lipoprotein; NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
comparisons between groups. The correlation between leptin and BMI was given by the Pearson correlation coefficient (r). The unpaired Student t test and the 2 test were used to compare the differences in demographic values between the groups. All analyses were two tailed and were conducted using a computer-based statistics software (SPSS for Windows 8.0, 1997, SPSS, Chicago, IL). A p value of less than 0.05 was accepted as statistically significant.
RESULTS The results of main parameters studied in groups are summarized in Table 2. The main predisposing factors for NASH were hyperlipidemia in 33 patients, obesity in 12 patients, and diabetes mellitus in 3 patients. Only 3 of the obese patients fit the definition of manifest obesity. All the diabetics and 7 of the obese patients had hyperlipidemia as well. Therefore, 38 patients had one or more predisposing factors for NASH, whereas the remaining 11 had no detectable predisposing cause. Four of the subjects in the control group were also obese, and 8 patients had hyperlipidemia. Despite the fact that about 75% of NASH patients were men in our group, since most of our patients were military personnel, sex distribution of patients in NASH and control group was not significantly different (2 ⫽ 2.91, p ⬎ 0.05). Serum leptin levels were found to be significantly higher in patients with NASH (6.62 ⫾ 0.7 ng/ml) as compared with both the control group (4.02 ⫾ 0.4 ng/ml, p ⫽ 0.011) and the chronic hepatitis group (4.24 ⫾ 1 ng/ml, p ⫽ 0.014; Fig. 1). There was no significant difference between mean levels of serum leptin of chronic hepatitis subjects and those of control subjects (p ⫽ 0.82). As expected, serum leptin levels were significantly higher in women subjects compared with those of men in all groups (p ⬍ 0.05; Table 3). For this reason, all comparisons were also made between men and women separately. Both men and women in the NASH group had higher mean serum leptin levels than did the men and women subjects of the control and chronic hepatitis groups (p ⬍ 0.05; Table 3). There was a positive correlation between BMI and leptin values for the combined groups (NASH, hepatitis, and con-
trol, r ⫽ 0.337, p ⫽ 0.012). When groups were analyzed separately, the correlation was also significant in the control group (r ⫽ 0.430, p ⫽ 0.018) and the hepatitis group (r ⫽ 0.441, p ⫽ 0.001), but not in the NASH group (r ⫽ 0.238, p ⫽ 0.1). Because the proportion of obese patients was rather low in the study population, for determining the relationship between obesity and leptin more clearly, the NASH patients were divided into two groups by using a BMI cutpoint score of 26. Thus, patients with a BMI of ⬎26 were defined as overweight. There were 20 patients with a BMI of ⬍26, and there were 29 patients with a BMI ⱖ26. The comparison of leptin values between these two groups was not significant, and the mean values were very close (Table 4). The serum leptin values were statistically higher in each of the two NASH groups (BMI ⬍ 26 or ⱖ26) than for those in the control group (p ⬍ 0.05 for both). These analyses revealed that the leptin values in NASH patients were higher in every way than were those of control subjects, without any significant effect for BMI. Histopathological examinations revealed that 29 of the
Figure 1. The serum leptin levels in nonalcoholic steatohepatitis patients, chronic hepatitis patients, and controls (bar graphic with 95% confidence interval for the mean).
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Table 3. Leptin and BMI Results of Groups According to Gender Men
Women
Variable
NASH
Hepatitis
Controls
Number (n) Leptin (ng/ml) BMI (kg/ml)
38 5.33 ⫾ 0.6 26.62 ⫾ 0.3
21 2.89 ⫾ 0.9 23.6 ⫾ 0.7
17 3.04 ⫾ 0.4 24.61 ⫾ 0.3
p
NASH
Hepatitis
Controls
p
S S
11 11.09 ⫾ 1.8 27.13 ⫾ 0.7
11 6.83 ⫾ 2.1 23.93 ⫾ 1.0
13 5.30 ⫾ 0.8 24.56 ⫾ 0.7
S S
S ⫽ significant at a p value of ⬍0.05.
NASH patients had grade 1 necroinflammatory activity, whereas 17 had grade 2 activity, and only 3 had grade 3 steatohepatitis. The mean level of serum leptin was 5.81 ⫾ 1.1 ng/ml in grade 1 patients and was 7.79 ⫾ 0.5 and 7.22 ⫾ 1.2 ng/ml in grade 2 and 3 patients, respectively. Because there were only three patients with grade 3 inflammation, they were included with the group 2 patients for statistical analyses. The mean serum leptin levels of the 20 patients with grade 2 and 3 hepatic inflammatory activity was significantly higher when compared with those of the 29 patients with grade 1 steatohepatitis (p ⫽ 0.018). We detected from the food frequency questionnaire that 42 of the patients with NASH were high fat consumers, whereas this was the case for only 7 of the controls. From the NASH patients with hyperlipidemia, 31 (93.3%) were also in the high fat consumer group. The C-peptide and insulin levels did not differ among groups (p ⬎ 0.05), but a positive correlation was found between serum leptin and insulin levels of NASH patients (r ⫽ 0.297, p ⫽ 0.038). The serum ALT, AST, glucose, total cholesterol, triglyceride, and low density lipoprotein (LDL) cholesterol levels were statistically higher in NASH patients than in controls (p ⬎ 0.05). However, none of these parameters displayed significant correlation with serum leptin levels in NASH patients.
DISCUSSION In this study, significantly high levels of serum leptin in NASH patients were found, which to our surprise, did not correlate with the BMI levels of the same group. On the other hand, a positive correlation between BMI and serum leptin levels was determined for the hepatitis subjects and Table 4. The Results of BMI, Leptin, and Main Biochemical Tests in NASH Patients With Normal Weight and Overweight Tests
Overweight (BMI ⱖ 26)
Normal Weight (BMI ⬍ 26)
p
Number (n) BMI (kg/m2) Leptin (ng/ml) C-peptide (ng/ml) Insulin (ng/ml) ALT (u/L) AST (u/L) Glucose (mg/dl) Total cholesterol (mg/dl) Triglyceride (mg/dl)
29 28.1 ⫾ 0.3 6.64 ⫾ 1.0 2.93 ⫾ 0.1 20.7 ⫾ 1.3 72.8 ⫾ 4.4 48.7 ⫾ 1.9 96.3 ⫾ 3.1 233.7 ⫾ 5.7 204.4 ⫾ 13.3
20 24.7 ⫾ 0.2 6.59 ⫾ 1.0 2.85 ⫾ 0.1 19.1 ⫾ 1.1 67.6 ⫾ 4.1 49.8 ⫾ 1.9 93.9 ⫾ 3.4 229.1 ⫾ 5.6 206.1 ⫾ 13.3
S NS NS NS NS NS NS NS NS
Data are mean ⫾ SEM. NS ⫽ nonsignificant; S ⫽ significant at a p value of ⬍0.05.
the control groups. The above results suggest no difference between normal and overweight NASH patients with regard to serum leptin levels. Our present findings contrast with previous reports suggesting higher levels of leptin in obese patients (2– 6). On the one hand, obesity is closely correlated with elevated levels of serum leptin; therefore, the majority of NASH patients may be expected to have high levels of serum leptin. On the other hand, the majority of our study patients displaying high levels of serum leptin were nonobese. Moreover, only three patients had a BMI above 30, which is the threshold for manifest obesity. This is probably because most of our patients were military personnel, among whom obesity is rarely seen. In fact, this situation may be considered to have been an advantage in displaying the role of leptin more clearly in NASH patients. It is an expected finding that leptin may increase in manifest obese patients with NASH. In such a patient group, it is difficult to explain whether serum leptin elevation is simply a consequence of obesity or whether it is really related to NASH. However, our study shows that serum leptin levels are increased in nonobese NASH patients and that it is impossible to explain serum leptin elevations in terms of obesity in the study group. Most of the NASH patients in this study exhibited hyperlipidemia. Serum total cholesterol, triglyceride, and LDL cholesterol levels of NASH patients were significantly elevated compared to controls. Lack of a familial hyperlipidemia history, absence of tendon xanthomas in examination, and no clinical disorders associated with secondary hyperlipidemia, except diabetes mellitus in three patients, suggested that in most of our patients, their lipoprotein concentrations are mainly influenced by their diet. It was determined from the food frequency questionnaire that nearly all the patients with hyperlipidemia were high-fat consumers in the study. This observation makes us consider the possibility that hyperlipidemia and a high-fat diet may induce important changes in leptin expression in adipose tissue without a significant increase in body fat content. In line with this suggestion, it was reported in a recent study that serum leptin levels were found elevated out of proportion to the BMI in patients with familial hyperlipidemia (17). The effect of a high-fat diet on serum leptin level is controversial in that a short-term high-fat diet may lower serum leptin levels (18, 19), but a long-term high-fat diet or habitual high-fat consumption is usually associated with increased serum leptin levels (20, 21). On the other hand, it is well known that food intake is the major promoting factor
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for leptin secretion. According to our results, like other previous studies, it can be suggested that a continuous high-fat diet and hyperlipidemia may result in high levels of serum leptin without a marked increase in BMI and manifest obesity. In a few recent studies, it has been demonstrated that serum leptin levels are increased in patients with liver cirrhosis (11–13). These studies confirm the suggestions that liver may play an important role on leptin metabolism and contribute to the regulation of circulating leptin levels. Neither the pathophysiological significance of this elevation nor the underlying exact mechanisms are clear at present. It has been suggested that hepatic failure in cirrhosis may cause this elevation by simply altering metabolism and clearance of leptin. In this study, no difference was determined between serum leptin levels of patients with chronic viral hepatitis and those of controls. Clinical, biochemical, or histopathological signs suggesting hepatic failure in hepatitis patients were not observed, and all the participants were in the uncirrhotic phase of the disease. At this point, our study confirms that leptin elevation in liver cirrhosis seems to be related to hepatic failure rather than to chronic inflammation or host immune response related to hepatic cell injury. On the other hand, the similar serum leptin levels in chronic viral hepatitis patients and controls point out that the increased serum leptin levels in NASH do not result simply from the liver damage. Whether serum leptin elevation has a metabolic effect in the development of steatosis and steatohepatitis is not clear at present. One of the important results of the study is that higher serum leptin levels were detected in patients with grade 2 and 3 steatohepatitis, compared with the grade 1 patients. This finding suggests that increased serum leptin level may be involved in the pathogenesis of steatohepatitis. However, this study is not enough to explain a possible relationship between leptin and hepatic fat accumulation and hepatic injury. It has been suggested that leptin may contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes so as to promote increased intracellular fatty acids (22). Moreover, in a later stage, leptin may cause hepatic steatosis to turn into steatohepatitis by amplifying selected proinflammatory responses (22). This explanation mainly is based on the effect of insulin on hepatic fat metabolism. In this study, an elevation of serum insulin level in NASH patients, in comparison to the control group, was not detected, but a positive significant correlation was determined between serum leptin and insulin levels, which supports the latter suggestion. It has been suggested that leptin substitution reverses insulin resistance and hepatic fat accumulation in some animal models with congenital lipodystrophy and leptin deficiency (23). However, this is not in question in the situation going on with increased leptin levels that has been shown to promote insulin resistance and probably intracellular fatty acids in hepatocytes (22). In summary, the data suggest that serum leptin level is
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increased in NASH patients inproportional to BMI. The high serum leptin levels may be secondary to hyperlipidemia in a significant number of these patients. Elevated serum leptin levels may therefore promote hepatic steatosis and steatohepatitis by insulin secretion and/or proinflammatory activation. However, further studies are needed to determine more clearly the role of leptin on hepatic steatosis and steatohepatitis.
Reprint requests and correspondence: Ahmet Uygun, M.D., GATA Lojmanları, Rieder Pasa, Apartment 5, Etlik/Ankara, Turkey. Received Sep. 14, 1999; accepted June 29, 2000.
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16. Pao EM, Cypel YS. Estimation of dietary intake. In: Ziegler EE, Filer LJ, eds. Present knowledge in nutrition, 7th ed. Washington, DC: ILSI Press, 1996:498 –507. 17. Jacobson MS, Yoon DJ, Frank GR. Serum leptin is elevated out of proportion to the body mass index in adolescent females with familial combined hyperlipidemia (FCH). Clin Pediatr 1999;38:49 –53. 18. Schrauwen P, Van Marken Lichtenbelt WD, Westerterp KR, et al. Effect of diet composition on leptin concentration in lean subjects. Metabolism 1997;46:420 – 4. 19. Weigle DS, Duell PB, Connor WE, et al. Effect of fasting, refeeding, and dietary fat restriction on plasma leptin levels. J Clin Endocrinol Metab 1997;82:561–5.
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20. Lovejoy JC, Windhauser MM, Rood JC, et al. Effect of a controlled high-fat versus low-fat diet on insulin sensitivity and leptin levels in African-American and Caucasian women. Metabolism 1998;47:1520 – 4. 21. Cooling J, Barth J, Blundell J. The high-fat phenotype: Is leptin involved in the adaptive response to a high fat (high energy) diet? Int J Obes Relat Metab Disord 1998;22: 1132–5. 22. Kaplan LM. Leptin, obesity and liver disease. Gastroenterology 1998;115:997–1001. 23. Shimomura I, Hammer RE, Ikemoto S, et al. Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy. Nature 1999;401:73– 6.